Injectable ready-to-use solutions containing an antitumor anthracycline glycoside

ABSTRACT

A sealed glass container containing therein a stable, injectable, sterile, pyrogen-free doxorubicin anti-tumor composition in a solution which consists essentially of a physiologically acceptable salt of doxorubicin dissolved in a physiologically acceptable solvent therefor, wherein said solution has not been reconstructed from a lyophilizate, and wherein said solution has a pH of from 2.5-3.5 and a concentration of said doxorubicin of from 0.1 to 100 mg/ml.

This application is a continuation of Ser. No. 07/341,249 filed Apr. 20,1989 which in turn is a continuation of Ser. No. 07/064.653 filed Jun.22, 1987, both now abandoned. Ser. No. 07/064,653 is acontinuation-in-part of Ser. No. 06/878,784 filed Jun. 26, 1986, nowabandoned which in turn was refiled as Ser. No. 07/385,999, filed Jul.27, 1989, now U.S. Pat. No. 4,946,831.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a stable, injectable, ready-to-usesolution of an antitumor anthracycline glycoside, e.g. doxorubicin(Adriamycin®), to a process for preparing such a solution, and providingthe same in a sealed container, and to a method for treating tumors bythe use of the ready-to-use solution.

2. Description of the Related Art

The anthracycline glycoside compounds are a well known class ofcompounds in the antineoplastic group of agents, of which doxorubicin isa typical, and the most widely used, representative: Doxorubicin.Anticancer Antibiotics, Federico Arcamone, 1981, Publ: Academic Press,New York, N.Y.; Adriamycin Review, EROTC International Symposium,Brussels, May, 1974, edited by M. Staquet, Publ. Eur. Press Medikon,Ghent, Belg.; Results of Adriamycin Therapy, Adriamycin Symposium atFrankfurt/Main 1974 edited by M. Ghione, J. Fetzer and H. Maier, publ.:Springer, New York, N.Y.

In the past, solutions of anthracycline glycosides have been preparedand the stability thereof has been studied. However, results of thesestudies have been inconsistent, and no clear parameters have emerged formaintenance of a stable anthracycline glycoside, e.g., doxorubicin,solution. Bosanquet, in a recent article entitled "Stability ofsolutions of antineoplastic agents during preparation and storage for invitro assays," (Cancer Chemother. Pharmacol. 1986, 17, 1-10) reviews thefield of stability studies, with particular emphasis on doxorubicin(Adriamycin®). He points out that "very little can be categoricallystated about the stability of adriamycin, and a very carefully designedstudy is urgently required to resolve these conflicting results."

At present, anthracycline glycoside antitumor drugs, in particular,e.g., doxorubicin, are solely available in the form of lyophilizedpreparations, which need to be reconstituted before administration.

Both the manufacturing and the reconstitution of such preparationsexpose the involved personnel (workers, pharmacists, medical personnel,nurses) to risks of contamination which are particularly serious due tothe toxicity of the antitumor substances.

Indeed, the Martindale Extra Pharmacopoeia 28th edition, page 175 leftcolumn, reports on adverse effects of antineoplastic drugs andrecommends that "They must be handled with great care and contact withskin and eyes avoided; they should not be inhaled. Care must be taken toavoid extravasation since pain and tissue damage may ensue".

Similarly, Scand. J. Work Environ Health vol. 10(2), pages 71-74 (1984),as well as articles in Chemistry Industry, Issue Jul. 4, 1983, page 488,and Drug-Topics-Medical-Economics-Co, Issue Feb. 7, 1983, page 99,report severe adverse effects observed in medical personnel exposed touse of cytostatic agents, including doxorubicin.

Even though the effect of long-term low-level exposure to such cytotoxicdrugs is not yet completely known, there is certainly a hazard for thosewho regularly prepare and administer these substances in view of thefact that they are known mutagens and carcinogens in animals andimplicated as carcinogens in man.

To administer a lyophilized preparation, double handling of the drug isrequired, the lyophilized cake having to be first reconstituted and thenadministered. Moreover, in some cases, the complete dissolution of thepowder may require prolonged shaking because of solubilization problems.Reconstitution of a lyophilized cake or powder can result in formationof aerosol droplets which can be inhaled or can come into contact withskin or mucous membranes of those handling the solution.

SUMMARY OF THE INVENTION

As the risks connected with the manufacturing and the reconstitution ofa lyophilized preparation would be highly reduced if a ready-to-usesolution of the drug were available, the present inventors havedeveloped a stable, therapeutically acceptable injectable solution of ananthracycline glycoside drug, e.g. doxorubicin, whose preparation andadministration does not require either lyophilization or reconstitution.

According to the present invention, there is provided a stable,injectable, sterile, pyrogen-free, anthracycline glycoside solutionwhich consists essentially of a physiologically acceptable salt of ananthracycline glycoside dissolved in a physiologically acceptablesolvent therefor, which has not been reconstituted from a lyophilizate,which has a pH of from 2.5 to 3.5 and which is preferably contained in asealed glass container.

BRIEF DESCRIPTION OF THE DRAWINGS

Drawing FIGS. 1 through 3 provide a pH profile for doxorubicn.HCLdegradation at 55° C. In FIG. 1 the profile is for sterile water, forFIG. 2 it is the profile in a 5% dextrose solution and in FIG. 3 aprofile for 0.9% saline solution.

Drawing FIGS. 4 through 6, represent a logarithmic plot residualdoxorubcin. HCL concentration versus time. In FIG. 4, the results arereported for sterile water solution, in FIG. 5 the results are for a0.9% sodium chloride solution and FIG. 6 represents the results obtainedin 5% dextrose.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A preferred pH range for the anthracycline glycoside solution of thisinvention is from 2.5 to 3.5. A more preferred range is from 2.7 to 3.5,and a particularly preferred range is from 2.7 to 3.3. A range of 2.7 upto about 3.0 may also be mentioned as a useful range.

The preferred anthracycline glycoside is doxorubicin hydrochloride. Itis known that doxorubicin hydrochloride is more stable in acidic thanneutral or basic solutions. See Analytical Profiles of Drug Substances,Vol. 9, Klaus Florey, ed. (Academic Press 1980). A number of stabilitystudies are summarized in Bosanquet, Cancer Chemother. Pharmacol. 17,1986, 1-10. However, these studies are inconsistent, in part because ofthe varying media used to make up the solutions and the methods used tomeasure stability. Taken as a whole, the prior art has not appreciatedwith any degree of certainty how to prepare a stable, injectabledoxorubicin solution, as a function of pH.

Martindale-The Extra Pharmacopeia 28th edition, 1828, on page 205,indicates that a 0.5% solution of doxorubicin hydrochloride and waterhas a pH of 3.8 to 6.5. Reconstitution of the commercial freeze driedformulation which is made of doxorubicin hydrochloride and lactose,leads to a solution having a pH in the range of between 4.5 and 6,containing doxorubicin at 2 milligrams per milliliter concentration andlactose, and additionally containing sodium chloride when saline is usedfor reconstitution. In order to lower the pH below that of reconstitutedsolutions, one must add an acid to lower the pH. In the past there waslittle motivation for a user of the drug to add an acid to lower the pH,since it was not recognized that the drug was actually more stable atpH's between about 2.5 and 3.5. According to the present invention, ithas been discovered that anthracycline glycosides, such as doxorubicinhydrochloride, are stable in the pH range disclosed herein inphysiologically acceptable media. The prior art did not recognizestability for an injectable doxorubicin solution in the particularnarrow pH range disclosed herein, so this pH range was not considered tobe a particularly useful range for administration of this drug.

Preferably the solution of the invention is provided in a sealedcontainer, especially one made of glass, either in a unit dosage form orin a multiple dosage form.

In addition to doxorubicin, other anthracycline glycosides include4'-epi-doxorubicin (i.e. epirubicin), 4'-desoxy-doxorubicin (i.e.esorubicin), 4'-desoxy-4'-iodo-doxorubicin, daunorubicin and4-demethoxydaunorubicin (i.e. idarubicin).

Any physiologically acceptable salt of the anthracycline glycoside maybe used for preparing the solution of the invention. Examples ofsuitable salts may be, for instance, the salts with mineral inorganicacids such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitricand the like, and the salts with certain organic acids such as acetic,succinic, tartaric, ascorbic, citric, glutamic, benzoic,methanesulfonic, ethanesulfonic and the like. The salt with hydrochloricacid is a particularly preferred salt, especially when the anthracyclineglycoside is doxorubicin.

Any solvent which is physiologically acceptable and which is able todissolve the anthracycline glycoside salt may be used. The solution ofthe invention may also contain one or more additional components such asa co-solubilizing agent (which may be the same as a solvent), a tonicityadjustment agent, a stabilizing agent and a preservative. Examples ofsolvents, co-solubilizing agents, tonicity adjustment agents,stabilizing agents and preservatives which can be used for thepreparation of the anthracycline glycoside solutions of the inventionare hereunder reported.

Suitable solvents and co-solubilizing agents may be, for instance,water; physiological saline; aliphatic amides, e.g.N,N-dimethylacetamide, N-hydroxy-2-ethyl-lactamide and the like;alcohols, e.g. ethanol, benzyl alcohol and the like; glycols andpolyalcohols, e.g. propyleneglycol, glycerin and the like; esters ofpolyalcohols, e.g. diacetine, triacetine and the like; polyglycols andpolyethers, e.g. polyethyleneglycol 400, propyleneglycol methylethersand the like; dioxolanes, e.g. isopropylidenglycerin and the like;dimethylisosorbide; pyrrolidone derivatives, e.g. 2-pyrrolidone,N-methyl-2-pyrrolidone, polyvinylpyrrolidone (co-solubilizing agentonly) and the like; polyoxyethylenated fatty alcohols, e.g., Brij® andthe like; esters of polyoxyethylenated fatty acids, e.g., Cremophor®,Myrj® and the like; polysorbates, e.g., Tweens®; polyoxyethylenederivatives of polypropyleneglycols, e.g., Pluronics®.

A particularly preferred co-solubilizing agent is polyvinylpyrrolidone.

Suitable tonicity adjustment agents may be, for instance,physiologically acceptable inorganic chlorides, e.g. sodium chloride;dextrose; lactose; mannitol; sorbitol and the like.

Preservatives suitable for physiological administration may be, forinstance, esters of parahydroxybenzoic acid (e.g., methyl, ethyl, propyland butyl esters, or mixtures of them), chlorocresol and the like.

Suitable stabilizing agents include monosaccharides (e.g., galactose,fructose, and fucose), disaccharides (e.g., lactose), polysaccharides(e.g., dextran), cyclic oligosaccharides (e.g., α-, β8-,γ-cyclodextrin), aliphatic polyols (e.g., mannitol, sorbitol, andthioglycerol), cyclic polyols (e.g.inositol) and organic solvents (e.g.,ethyl alcohol and glycerol). These may be included in concentrations offrom about 0.25-10% w/v, preferably 0.5-5% w/v in the solution.

The above mentioned solvents and co-solubilizing agents, tonicityadjustment agents, stabilizing agents and preservatives can be usedalone or as a mixture of two or more of them.

Examples of preferred solvents are water, ethanol, polyethylene glycoland dimethylacetamide as well as mixtures in various proportions ofthese solvents. Water is a particularly preferred solvent. Othersolvents giving good results in terms of stability are 0.9% sodiumchloride solution (i.e., physiological saline), and, especially, 5%dextrose solution, 5% mannitol solution and 5% sorbitol solution, i.e.,aqueous solutions containing approximately 5% of, respectively,dextrose, mannitol or sorbitol. Small variations (±2-3%) of theseadditional ingredients also fall within the scope of the presentinvention.

To adjust the pH within the range of from 2.5 to about 3.5, aphysiologically acceptable acid is added to the solution of theanthracycline glycoside. The acid should be a physiologically acceptableacid, e.g., an inorganic mineral acid such as hydrochloric, hydrobromic,sulfuric, phosphoric, nitric and the like, or an organic acid such asacetic, succinic, tartaric, ascorbic, citric, glutamic, benzoic,methanesulphonic, ethanesulfonic and the like, or also an acidicphysiologically acceptable buffer solution, e.g., a chloride buffer, anacetate buffer, a phosphate buffer and the like.

In the solutions of the invention the concentration of the anthracyclineglycoside may vary within broad ranges, preferably from 0.1 mg/ml to 100mg/ml, in particular from 0.1 mg/ml to 50 mg/ml, most preferably from 1mg/ml to 20 mg/ml.

The preferred ranges of concentration may be slightly different fordifferent anthracycline glycosides. Thus, for example, preferredconcentrations for doxorubicin are from about 2 mg/ml to about 50 mg/ml,preferably from 2 mg/ml to 20 mg/ml, particularly appropriate valuesbeing 2 mg/ml and 5 mg/ml. Similar concentrations are preferred also for4'-epi-doxorubicin, 4'-desoxy-doxorubicin and4'-desoxy-4'-iodo-doxorubicin. Preferred ranges of concentration fordaunorubicin and 4-demethoxydaunorubicin are from 0.1 mg/ml to 50 mg/ml,preferably from 1 mg/ml to 20 mg/ml, concentrations of 1 mg/ml and 5mg/ml being particularly appropriate.

Suitable packaging for the anthracycline glycoside solutions may be allapproved containers intended for parenteral use, such as plastic andglass containers, ready-to-use syringes and the like. Preferably thecontainer is a sealed glass container, e.g. a vial or an ampoule. Ahermetically sealed glass vial is particularly preferred.

According to a particularly preferred feature of the invention, there isprovided, in a sealed glass container, a sterile, pyrogen-free,injectable doxorubicin solution which consists essentially of aphysiologically acceptable salt of doxorubicin dissolved in aphysiologically acceptable solvent therefor, which has not beenreconstituted from a lyophilizate and which has a pH of from 2.5 to 3.5.

In the above indicated preferred feature of the invention thephysiologically acceptable salt of doxorubicin may be, e.g. the saltwith a mineral inorganic acid such as hydrochloric, hydrobromic,sulfuric, phosphoric, nitric and the like, or the salt with an organicacid such as acetic, succinic, tartaric, ascorbic, citric, glutamic,benzoic, methanesulfonic, ethanesulfonic and the like. The hydrochloridesalt is a particularly preferred salt.

For the solution hereabove indicated as a preferred feature of theinvention suitable solvents, co-solubilizing agents, tonicity adjustmentagents, stabilizing agents and preservatives may be the same as thosepreviously recited in this specification. Water is a particularlypreferred solvent.

Also, the physiologically acceptable acid which is added to adjust thepH to from 2.5 to about 3.5 may be one of those previously specified.When it is desired to adjust the pH of the above said preferred solutionto a value of from 2.5 to about 3.5, phydrochloric acid is an especiallypreferred acid. Preferred pH values for the above said preferredsolutions of the invention are from about 2.7 to about 3.3.

Though the concentration of doxorubicin in the above preferred featuremay vary within the broad range from 0.1 mg/ml to 100 mg/ml, preferredconcentrations are from 2 mg/ml to 50 mg/ml, most preferably from 2mg/ml to 20 mg/ml: examples of especially preferred concentrations ofdoxorubicin are 2 mg/ml and 5 mg/ml.

The invention also provides a process for producing a sterile,pyrogen-free anthracycline glycoside solution with a pH of from 2.5 to3.5 which process comprises dissolving a physiologically acceptable saltof the anthracycline glycoside, which salt is not in the form of alyophilizate, in a physiologically acceptable solvent therefor; adding aphysiologically acceptable acid or buffer to adjust the pH within thesaid range as desired; and passing the resulting solution through asterilizing filter.

One or more additional components such as co-solubilizing agents,tonicity adjustment agents, stabilizing agents and preservatives, forinstance of the kind previously specified, may be added to the solutionprior to passing the solution through the sterilizing filter.

With the solutions of the invention it is possible to obtaincompositions having a very high concentration of the anthracyclineglycoside active substance even at 50 mg/ml and more. This constitutes agreat advantage over the presently available lyophilized preparationswherein high concentrations of anthracycline glycoside can only beobtained with difficulty because of solubilization problems encounteredin reconstitution, mainly with saline. The presence of the excipient,e.g. lactose, in the lyophilized cake, and its generally high proportionin respect of the active substance, even up to 5 parts of excipient perpart of active substance, has a negative effect on solubilization sothat difficulties may arise in obtaining dissolution of the lyophilizedcake, especially for concentrations of anthracycline glycoside higherthan 2 mg/ml.

The solutions of the invention are characterized by a good stability.Solutions in various solvents and with different pH's and concentrationshave been found to be stable for long periods at temperatures acceptedfor the storage of pharmaceutical preparations. This is illustrated inthe Examples which follow.

Due to the well known anti-tumor activity of the anthracycline glycosideactive drug substance, the pharmaceutical compositions of the inventionare useful for treating tumors in both human and animal hosts. Examplesof tumors that can be treated are, for instance, sarcomas, includingosteogenic and soft tissue sarcomas, carcinomas, e.g., breast-, lung-,bladder-, thyroid-, prostate- and ovarian carcinoma, lymphomas,including Hodgkin and non-Hodgkin lymphomas, neuroblastoma, melanoma,myeloma, Wilms tumor, and leukemias, including acute lymphoblasticleukemia and acute myeloblastic leukemia. See Kirk-Othmer's Encyclopediaof Chemical Technology, Volume 5, pages 478-479 (1979).

Examples of specific tumors that can be treated are Moloney SarcomaVirus, Sarcoma 180 Ascites, solid Sarcoma 180, gross transplantableleukemia, L 1210 leukemia and lymphocytic P 388 leukemia.

Thus, according to the invention there is also provided a method ofinhibiting the growth of a tumor, in particular one of those indicatedabove, which comprises administering to a host suffering from said tumoran injectable solution according to the invention containing the activedrug substance in an amount sufficient to inhibit the growth of saidtumor.

The injectable solutions of the invention are administered by rapidintravenous injection or infusion according to a variety of possibledose schedules. A suitable dose schedule for doxorubicin may be, forexample, of 60 to 75 mg of active drug substance per m² of body surfacegiven as a single rapid infusion and repeated at 21 days; an alternativeschedule may be of 30 mg/m² per day by intravenous route for 3 days,every 28 days. Suitable dosages for 4'-epi-doxorubicin and4'-desoxy-doxorubicin may be, for instance, of 75 to 90 mg/m² given in asingle infusion to be repeated at 21 days, and similar dosages may beuseful also for 4'-desoxy-4'-iodo-doxorubicin.

Idarubicin, i.e. 4-demethoxy-daunorubicin, may be administeredintravenously at a single dose of 13-15 mg/m² every 21 days in thetreatment of solid tumors, while in the treatment of leukemias apreferred dose schedule is, e.g., of 10-12 mg/m² day by intravenousroute for 3 days, to be repeated evey 15-21 days; similar dosages may befollowed also for daunorubicin.

Examples

The invention now being generally described, the same will be betterunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting of the invention or any embodiment thereof, unless specified.

With reference to the first three examples, the stability controls onthe ready-to-use solutions were carried out by means of high performanceliquid chromatography (HPLC), at the following experimental conditions:

    ______________________________________                                        Liquid chromatograph                                                                             Varian model 5010                                          Spectrophotometric detector                                                                      Knauer model 8700                                          Integrating recorder                                                                             Varian model CDS 401                                       Injection valve    Rheodyne model 7125                                                           fitted with a 10 mcl                                                          sample loop                                                Chromatographic column                                                                           Waters μ-Bondapak C18                                                      (length = 300 mm; inner                                                       diameter = 3.9 mm;                                                            average particle size =                                                       10 mcm)                                                    Column temperature ambient (about 22° C. ±                                             2° C.)                                              Mobile phase       water : acetonitrile                                                          (69:31 v/v) adjusted to                                                       pH 2 with phosphoric                                                          acid, filtered                                                                (sintered glass filter,                                                       1 mcm or finer                                                                porosity) and deaerated                                    Mobile phase flow rate                                                                           1.5 ml/min                                                 Analytical wavelength                                                                            254 ± 1 nm                                              Integrating recorder                                                                             512                                                        sensitivity                                                                   Chart speed        1 cm/min                                                   ______________________________________                                    

At these conditions, the peak of the anthracycline glycoside showed aretention time of about 6 minutes.

The obtained results are reported in Tables accompanying Examples 1-3.

The extrapolation of the analytical data in order to determine the timewhen the 90% of the initial assay could be expected (t₉₀ value) was madefollowing an Arrhenius plot.

This procedure of analytical data treatment is well known and widelyused and described in the art: see e.g., Chemical Stability ofPharmaceuticals, Kennet A. Connors, Gordon L. Amidon, Lloyd Kennon,Publ. John Wiley and Sons, New York, N.Y., 1979.

The term "teflon" recurring in the examples refers to "Teflon™".

EXAMPLE 1

    ______________________________________                                        Composition     for 80 vials (for 1 vial)                                     ______________________________________                                        Doxorubicin.HCl 0.8 g        (10 mg)                                          Water for injections                                                                          0.4 l         (5 ml)                                          q.s. to                                                                       ______________________________________                                    

Doxorubicin.HCl (0.80 g) was dissolved in 90 percent of the amount ofwater for injections, de-aerated by nitrogen bubbling. The pH of thesolution was not adjusted. Further de-aerated water for injections wasthen added to bring the solution to its final volume (0.40 1).

The solution was filtered through a 0.22 μ microporous membrane undernitrogen pressure. Volumes of 5 ml of the solution were distributed intotype I-colorless glass vials having 5/7 ml capacity. The vials were thenclosed with chlorobutyl teflon-faced rubber stoppers and sealed withaluminum caps.

The stability of the solutions in the vials was tested. The vials werestored at temperatures of 55° C., 45° C. and 35° C. (acceleratedstability controls) and at 4° C. for up to 3 weeks (55° C.), 4 weeks(45° C. and 35° C.) and 12 weeks (4° C.).

The stability data obtained, using high performance liquidchromatography (HPLC) for the determination of potency, are reported inthe following Table 1:

                                      TABLE 1                                     __________________________________________________________________________           INITIAL VALUES                                                                Concentration:                                                                           1.994 mg/ml                                                                          pH = 5.2                                                    Relative % Assay:                                                                       100.0                                                        TEMPERATURE                                                                   4° C. 35° C.                                                                         45° C.                                                                         55° C.                                    TIME Conc.                                                                             Rel. %                                                                            Conc.                                                                             Rel %                                                                             Conc.                                                                             Rel. %                                                                            Conc.                                                                             Rel %                                        (weeks)                                                                            mg/ml                                                                             Assay                                                                             mg/ml                                                                             Assay                                                                             mg/ml                                                                             Assay                                                                             mg/ml                                                                             Assay                                        __________________________________________________________________________    1    1.992                                                                             99.9                                                                              1.917                                                                             96.1                                                                              1.768                                                                             88.7                                                                              1.493                                                                             75.0                                         2            1.843                                                                             92.4                                                                              1.618                                                                             81.1                                                                              1.166                                                                             58.5                                         3            1.774                                                                             89.0                                                                              1.506                                                                             75.5                                                                              0.830                                                                             41.6                                         4    1.974                                                                             99.0                                                                              1.720                                                                             86.3                                                                              1.393                                                                             69.9                                                 12   1.980                                                                             99.3                                                                 __________________________________________________________________________     t.sub.90 (days) extrapolated according to Arrhenius equation:                 t.sub.90 at 4° C. = 815 days                                           t.sub.90 at 8° C. = 480 days                                      

Similar stability data can be observed also for analogous solutionscontaining either doxorubicin hydrochloride at 5 mg/ml concentration, or4'-epidoxorubicin, 4'-desoxy-doxorubicin, 4'-desoxy-4'-iododoxorubicin,daunorubicinn or 4-demethoxy-daunorubicin, as hydrochloride salts, atboth 2 mg/ml and 5 mg/ml concentration.

EXAMPLE 2

    ______________________________________                                        Composition      for 80 vials                                                                              (for 1 vial)                                     ______________________________________                                        Doxorubicin.HCl  0.8 g       (10 mg)                                          Hydrochloric acid 0.1N                                                                         pH = 3      (pH = 3)                                         q.s. to                                                                       Water for injections                                                                           0.4 l        (5 ml)                                          q.s. to                                                                       ______________________________________                                    

Doxorubicin.HCl (0.8 g) was dissolved in 90 percent of the amount ofwater for injections, de-aerated by nitrogen bubbling. The hydrochloricacid was then added dropwise to adjust the pH of the solution to 3.Further de-aerated water for injections was then added to bring thesolution to its final volume (0.4 1).

The solution was filtered through a 0.22μ microporous membrane undernitrogen pressure. Volumes of 5 ml of the solution were distributed intotype I-colorless glass vials having 5/7 ml capacity. The vials were thenclosed with chlorobutyl teflon-faced rubber stoppers and sealed withaluminum caps.

The stability of the solutions in the vials was tested. The vials werestored at temperatures of 55° C., 45° C. and 35° C. (acceleratedstability controls) and at 4° C. for up to 3 weeks (55° C.), 4 weeks(45° C. and 35° C.) and 12 weeks (4° C.).

The stability data obtained, using high performance liquidchromatography (HPLC) for the determination of potency, are reported inthe following Table 2:

                                      TABLE 2                                     __________________________________________________________________________           INITIAL VALUES                                                                Concentration:                                                                           1.992 mg/ml                                                                          pH = 3.0                                                    Relative % Assay:                                                                       100.0                                                        TEMPERATURE                                                                   4° C. 35° C.                                                                         45° C.                                                                         55° C.                                    TIME Conc.                                                                             Rel. %                                                                            Conc.                                                                             Rel %                                                                             Conc.                                                                             Rel. %                                                                            Conc.                                                                             Rel %                                        (weeks)                                                                            mg/ml                                                                             Assay                                                                             mg/ml                                                                             Assay                                                                             mg/ml                                                                             Assay                                                                             mg/ml                                                                             Assay                                        __________________________________________________________________________    1    1.995                                                                             100.2                                                                             1.952                                                                             98.0                                                                              1.919                                                                             96.3                                                                              1.493                                                                             75.0                                         2            1.889                                                                             94.8                                                                              1.851                                                                             92.9                                                                              1.036                                                                             51.9                                         3            1.876                                                                             94.2                                                                              1.565                                                                             78.6                                                                              0.730                                                                             36.7                                         4    1.979                                                                             99.4                                                                              1.808                                                                             90.8                                                                              1.393                                                                             69.9                                                 12   1.972                                                                             99.0                                                                 __________________________________________________________________________     t.sub.90 (days) extrapolated according to Arrhenius equation:                 t.sub.90 at 4° C. = 3970 days                                          t.sub.90 at 8° C. = 2000 days                                     

Similar stability data can be observed also for analogous solutionscontaining either doxorubicin hydrochloride at 5 mg/ml concentration, or4'-epi-doxorubicin, 4'-desoxy-doxorubicin,4'-desoxy-4'-iodo-doxorubicin, daunorubicin or 4-demethoxy-daunorubicin,as hydrochloride salts, at both 2 mg/ml and 5 mg/ml concentration.

EXAMPLE 3

    ______________________________________                                        Composition      for 80 vials                                                                              (for 1 vial)                                     ______________________________________                                        Doxorubicin.HCl  8.0 g       (100 mg)                                         Hydrochloric acid 0.1N                                                                         pH = 3      (pH = 3)                                         q.s. to                                                                       Water injections 0.4 l        (5 ml)                                          q.s. to                                                                       ______________________________________                                    

Doxorubicin.HCl (8.0 g) was dissolved in 90 percent of the amount ofwater for injections, deaerated by nitrogen bubbling. The hydrochloricacid was then added dropwise to adjust the pH of the solution 3. Furtherde-aerated water for injections was then added to bring the solution toits final volume (0.4 1).

The solution was filtered through a 0.22μ microporous membrane undernitrogen pressure. Volumes of 5 ml of the solution were distributed intotype I-colorless glass vials having 5/7 ml capacity. The vials were thenclosed with chlorobutyl teflon-faced rubber stoppers and sealed withaluminum caps.

The stability of the solutions in the vials was tested. The vials werestored at temperatures of 55° C., 45° C. and 35° C. (acceleratedstability controls) and at 4° C. for up to 3 weeks (55° C.), 4 weeks(45° C. and 35° C.) and 12 weeks (4° C.).

The stability data obtained, using high performance liquidchromatography (HPLC) for the determination of potency, are reported inthe following Table 3:

                                      TABLE 3                                     __________________________________________________________________________           INITIAL VALUES                                                                Concentration:                                                                           20.06 mg/ml                                                                          pH = 2.95                                                   Relative % Assay:                                                                       100.0                                                        TEMPERATURE                                                                   4° C. 35° C.                                                                         45° C.                                                                         55° C.                                    TIME Conc.                                                                             Rel. %                                                                            Conc.                                                                             Rel %                                                                             Conc.                                                                             Rel. %                                                                            Conc.                                                                             Rel %                                        (weeks)                                                                            mg/ml                                                                             Assay                                                                             mg/ml                                                                             Assay                                                                             mg/ml                                                                             Assay                                                                             mg/ml                                                                             Assay                                        __________________________________________________________________________    1    20.06                                                                             100.0                                                                             19.56                                                                             97.5                                                                              17.84                                                                             88.9                                                                              12.31                                                                             61.4                                         2            18.87                                                                             94.1                                                                              15.61                                                                             77.8                                                                              7.09                                                                              35.3                                         3            18.24                                                                             90.9                                                                              13.41                                                                             66.8                                                                              3.13                                                                              15.6                                         4    19.91                                                                             99.2                                                                              17.51                                                                             87.3                                                                              11.07                                                                             55.2                                                 12   19.80                                                                             98.7                                                                 __________________________________________________________________________     t.sub.90 (days) extrapolated according to Arrhenius equation:                 t.sub.90 at 4° C. = 3700 days                                          t.sub.90 at 8° C. = 1780 days                                     

Similar stability data can be observed for analogous solutionscontaining 4'-epi-doxorubicin or 4'-desoxy-doxorubicin, as hydrochloridesalts, at the same 20 mg/ml concentration.

The following examples regarding stability profile and shelf-lifeforecasts were carried out under accelerated temperature conditions on5.0 ml of 2 mg/ml doxorubicin.HCl solutions in a container-closuresystem consisting of: glass type I, 8 ml top capacity vial; teflon-facedchlorobutyl rubber bung; aluminum seal.

pH-stability profile at 55° C. of doxorubicin.HCl solutions in sterilewater 5% dextrose, 0.9% saline

2 mg/ml doxorubicin.HCl solutions were prepared in the following I=0.05buffers: a) glycine.HCl pH 2.0, 2.5 and 3.0; b) formate pH 3.5; c)acetate pH 4.0, 5.0 and 5.5.

5.0 ml of each solution in glass vials were stored at 55° C. andanalyzed at prefixed times (up to 120 hours) for doxorubicin.HCl assayand pH.

Tables 4, 5 and 6 give the doxorubicin.HCl residual concentration andpercent stability at 55° C., at different pH's and times of storage forsterile water, 5% dextrose and 0.9% saline solutions, respectively.

The doxorubicin.HCl assays are the mean of three independentdeterminations performed by the USP HPLC method (USP XXI). At each pHvalue, the pseudo-first order rate constants (K_(obs)) for thedegradation were calculated by linear regression analysis of the naturallogarithm of the residual concentration of doxorubicin.HCl (|Dx|_(t))versus time as depicted by the following equation:

    ln|Dx|.sub.t =ln|Dx|-K.sub.obs ·t

J. Thuro Carstensen, Theory of Pharmaceutical Systems, Volume 1/GeneralPrinciples, page 172, Academic Press, New York and London 1972

Kenneth A. Connors, Gordon L. Amidon, Lloyd Kennon, Chemical Stabilityof Pharmaceuticals, chapter 2, John Wiley and Sons, New York 1979

Arthur Osol, Remington's Pharmaceutical Sciences, 16th Edition, chapter18, Mack Publishing Company, Easton, Pa. 1980

Valentino J. Stella, Chemical and Physical Bases Determining theInstability and Incompatibility of Formulated Injectable Drugs, Journalof Parenteral Sciences & Technology, Jul.-Aug. 1986, page 142

Tables 7, 8 and 9 give the observed rate constants (K_(obs)) for thedegradation kinetics of doxorubicin.HCl at 55° C. and at different pH'sfor sterile water, 5% dextrose and 0.9% saline solutions, respectively.

Drawing FIGS. 1 through 3 show the K_(obs) -pH profile for thedoxorubicin.HCl degradation at 55° C. in the above mentioned media. Thedata in Tables 4-9 and the FIGS. 1-3 evidence that the 2 mg/mldoxorubicin.HCl solutions show at 55° C. the maximum stability in the pHrange about 3.0-3.5 (±0.2, e.g., 2.8, 3.2 and 3.3, 3.7) for all thethree media tested. The range of from 2.5 to 3.0 is also a range ofnotable stability.

A common behavior as to stability is thus evidenced for aqueoussolutions in general, since no practical differences in stability areobserved when going from sterile water as such to sterile watercontaining a tonicity adjustment agent, either ionic, such as, e.g.,sodium chloride, or non-ionic, such as, e.g., dextrose.

                                      TABLE 4                                     __________________________________________________________________________    Accelerated (55° C.) stability data of 2 mg/ml doxorubicin.HCl         solutions in                                                                  sterile water at various pHs                                                                     Time (hours)                                               Buffers                                                                              Tests       0   8   16  24  48  72  120                                __________________________________________________________________________    pH 2.0 Doxorubicin.HCl assay                                                  glycine-HCl                                                                          mg/ml       2.022                                                                             1.892                                                                             1.669                                                                             1.554                                                                             1.145                                                                             0.801                                         % stability 100.0                                                                             93.6                                                                              82.6                                                                              76.9                                                                              56.6                                                                              39.6                                          pH          2.00                                                                              2.01                                                                              2.02                                                                              2.01                                                                              2.01                                                                              2.02                                   pH 2.5 Doxorubicin.HCl assay                                                  glycine-HCl                                                                          mg/ml       1.992                                                                             1.926                                                                             1.835                                                                             1.718                                                                             1.557                                                                             1.00                                          % stability 100.0                                                                             96.7                                                                              92.1                                                                              86.2                                                                              78.2                                                                              50.2                                          pH          2.51                                                                              2.50                                                                              2.50                                                                              2.52                                                                              2.51                                                                              2.52                                   pH 3.0 Doxorubicin.HCl assay                                                  glycine-HCl                                                                          mg/ml       2.003                                                                             1.958                                                                             1.881                                                                             1.831                                                                             1.696                                                                             1.525                                                                             1.258                                     % stability 100.0                                                                             97.8                                                                              93.9                                                                              91.4                                                                              84.7                                                                              76.1                                                                              62.8                                      pH          3.00                                                                              3.03                                                                              3.02                                                                              3.02                                                                              3.01                                                                              3.02                                                                              3.00                               pH 3.5 Doxorubicin.HCl assay                                                  formate                                                                              mg/ml       2.035                                                                             1.950                                                                             1.887                                                                             1.840                                                                             1.650                                                                             1.538                                                                             1.241                                     % stability 100.0                                                                             95.8                                                                              92.7                                                                              90.4                                                                              81.1                                                                              75.6                                                                              61.0                                      pH          3.51                                                                              3.51                                                                              3.51                                                                              3.51                                                                              3.52                                                                              3.52                                                                              3.51                               pH 4.0 Doxorubicin.HCl assay                                                  acetate                                                                              mg/ml       2.032                                                                             1.788                                                                             1.681                                                                             1.561                                                                             1.167                                             % stability 100.0                                                                             88.0                                                                              82.7                                                                              76.8                                                                              57.4                                              pH          4.00                                                                              4.00                                                                              4.04                                                                              4.02                                                                              4.02                                       pH 5.0 Doxorubicin.HCl assay                                                  acetate                                                                              mg/ml       2.019                                                                             1.823                                                                             1.688                                                                             1.512                                                                             1.060                                             % stability 100.0                                                                             90.3                                                                              83.6                                                                              74.9                                                                              52.5                                              pH          5.03                                                                              5.05                                                                              5.04                                                                              5.04                                                                              5.05                                       pH 5.5 Doxorubicin.HCl assay                                                  acetate                                                                              mg/ml       2.047                                                                             1.808                                                                             1.427                                                                             1.228                                                                             0.903                                             % stability 100.0                                                                             88.3                                                                              69.7                                                                              60.0                                                                              44.1                                              pH          5.50                                                                              5.53                                                                              5.53                                                                              5.54                                                                              5.56                                       __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Accelerated (55° C.) stability data of 2 mg/ml doxorubicin.HCl         solutions in 5% dextrose                                                      at various pHs                                                                                   Time (hours)                                               Buffers                                                                              Tests       0   8   16  24  34  48  72  96  120                        __________________________________________________________________________    pH 2.0 Doxorubicin.HCl assay                                                  glycine-HCl                                                                          mg/ml       1.993                                                                             1.851                                                                             1.683                                                                             1.513                                                                             1.361                                                                             1.078                                                                             0.765                                     % stability 100.0                                                                             92.8                                                                              84.4                                                                              75.9                                                                              68.3                                                                              54.1                                                                              38.4                                      pH          2.14                                                                              2.13                                                                              2.14                                                                              2.15                                                                              2.18                                                                              2.21                                                                              2.16                               pH 2.5 Doxorubicin.HCl assay                                                  glycine-HCl                                                                          mg/ml       1.967                                                                             1.897                                                                             1.822                                                                             1.760                                                                             1.682                                                                             1.499                                                                             1.305                                     % stability 100.0                                                                             96.4                                                                              92.6                                                                              89.5                                                                              85.5                                                                              76.2                                                                              66.3                                      pH          2.56                                                                              2.56                                                                              2.56                                                                              2.58                                                                              2.60                                                                              1.56                                                                              2.61                               pH 3.0 Doxorubicin.HCl assay                                                  glycine-HCl                                                                          mg/ml       1.975   1.908                                                                             1.832   1.645                                                                             1.508                                                                             1.344                                                                             1.206                             % stability 100.0   96.6                                                                              92.7    83.3                                                                              76.4                                                                              68.0                                                                              61.1                              pH          3.04    3.05                                                                              3.05    3.06                                                                              3.00                                                                              3.13                                                                              3.10                       pH 3.5 Doxorubicin.HCl assay                                                  formate                                                                              mg/ml       1.983   1.897                                                                             1.858   1.622                                                                             1.324                                                                             1.222                                 % stability 100.0   95.7                                                                              93.7    81.8                                                                              66.8                                                                              61.6                                  pH          3.58    3.59                                                                              3.60    3.63                                                                              3.60                                                                              3.63                           pH 4.0 Doxorubicin.HCl assay                                                  acetate                                                                              mg/ml       2.003                                                                             1.913                                                                             1.716                                                                             1.665                                                                             1.487                                                                             1.312                                                                             1.081                                     % stability 100.0                                                                             95.5                                                                              85.6                                                                              83.1                                                                              74.2                                                                              65.5                                                                              53.9                                      pH          4.10                                                                              4.10                                                                              4.11                                                                              4.11                                                                              4.16                                                                              4.15                                                                              4.12                               pH 5.0 Doxorubicin.HCl assay                                                  acetate                                                                              mg/ml       2.012                                                                             1.906                                                                             1.673                                                                             1.608                                                                             1.416                                                                             1.163                                         % stability 100.0                                                                             94.7                                                                              83.2                                                                              79.9                                                                              70.4                                                                              57.8                                          pH          5.06                                                                              5.06                                                                              5.06                                                                              5.06                                                                              5.07                                                                              5.04                                   pH 5.5 Doxorubicin.HCl assay                                                  acetate                                                                              mg/ml       1.991                                                                             1.841                                                                             1.470                                                                             1.246                                                                             1.091                                             % stability 100.0                                                                             92.5                                                                              73.8                                                                              62.6                                                                              54.8                                              pH          5.56                                                                              5.54                                                                              5.48                                                                              5.50                                                                              5.46                                       __________________________________________________________________________

                                      TABLE 6                                     __________________________________________________________________________    Accelerated (55° C.) stability data of 2 mg/ml doxorubicin.HCl         solutions in 0.9% saline                                                      at various pHs                                                                                 Time (hours)                                                 Buffers                                                                            Tests       0   4   8   16  24  34  48  72  96  120                      __________________________________________________________________________    pH 2.0                                                                             Doxorubicin.HCl assay                                                    glycine-                                                                           mg/ml       1.998   1.857                                                                             1.580                                                                             1.397                                                                             1.231                                                                             0.931                                                                             0.701                                 % stability 100.0   92.9                                                                              79.1                                                                              69.9                                                                              61.6                                                                              46.6                                                                              35.1                                  pH          2.16    2.16                                                                              2.18                                                                              2.16                                                                              2.22                                                                              2.20                                                                              2.19                             pH 2.5                                                                             Doxorubicin.HCl assay                                                    glycine-                                                                           mg/ml       1.946   1.875                                                                             1.670                                                                             1.602                                                                             1.368                                                                             1.132                                HCl  % stability 100.0   96.3                                                                              85.8                                                                              82.3                                                                              70.3                                                                              58.1                                      pH          2.59    2.59                                                                              2.59                                                                              2.58                                                                              2.62                                                                              2.62                                 pH 3.0                                                                             Doxorubicin.HCl assay                                                    glycine-                                                                           mg/ml       1.994       1.818                                                                             1.771   1.571                                                                             1.375                                                                             1.205                                                                             1.003                    HCl  % stability 100.0       91.2                                                                              88.8    78.8                                                                              69.0                                                                              60.4                                                                              50.3                          pH          3.06        3.07                                                                              3.07    3.08                                                                              3.13                                                                              3.14                                                                              3.12                     pH 3.5                                                                             Doxorubicin.HCl assay                                                    formate                                                                            mg/ml       1.997       1.824                                                                             1.742   1.543                                                                             1.323                                                                             1.176                                                                             0.919                         % stability 100.0       91.4                                                                              87.2    77.3                                                                              66.2                                                                              58.9                                                                              46.0                          pH          3.58        3.56                                                                              3.56    3.66                                                                              3.61                                                                              3.64                                                                              3.63                     pH 4.0                                                                             Doxorubicin.HCl assay                                                    acetate                                                                            mg/ml       1.972                                                                             1.885                                                                             1.828                                                                             1.653                                                                             1.594                                             % stability 100.0                                                                             95.6                                                                              92.7                                                                              83.8                                                                              80.8                                              pH          4.10                                                                              4.10                                                                              4.10                                                                              4.10                                                                              4.11                                         pH 5.0                                                                             Doxorubicin.HCl assay                                                    acetate                                                                            mg/ml       1.979   1.732                                                                             1.469                                                                             1.442                                                                             1.278                                         % stability 100.0   87.5                                                                              74.2                                                                              72.8                                                                              64.6                                          pH          5.04    5.06                                                                              5.04                                                                              5.05                                                                              5.05                                     pH 5.5                                                                             Doxorubicin.HCl assay                                                    acetate                                                                            mg/ml       2.023   1.847                                                                             1.548                                                                             1.330                                             % stability 100.0   91.3                                                                              76.5                                                                              65.7                                              pH          5.58    5.56                                                                              5.55                                                                              5.53                                         __________________________________________________________________________

                  TABLE 7                                                         ______________________________________                                        K.sub.obs values (1/days) for the degradation of doxorubicin.HCl              2 mg/ml solutions in sterile water at various pHs at 55° C.            Buffer     pH      K.sub.obs × 10.sup.3                                                              95% confidence limits                            ______________________________________                                        Glycine-HCl                                                                              2.0     309.5     ±12.6                                         (I = 0.05)                                                                    Glycine-HCl                                                                              2.5     138.3      ±0.6                                         (I = 0.05)                                                                    Glycine-HCl                                                                              3.0      93.1      ±4.6                                         (I = 0.05)                                                                    Formate    3.5      96.7      ±4.4                                         (I = 0.05)                                                                    Acetate    4.0     269.8     ±18.7                                         (I = 0.05)                                                                    Acetate    5.0     322.6     ±19.2                                         (I = 0.05)                                                                    Acetate    5.5     415.4     ±45.7                                         (I = 0.05)                                                                    ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        K.sub.obs values (1/days) for the degradation of doxorubicin.HCl              2 mg/ml solutions in 5% dextrose at various pHs at 55° C.              Buffer     pH      K.sub.obs × 10.sup.3                                                              95% confidence limits                            ______________________________________                                        Glycine-HCl                                                                              2.0     323.8     ±17.2                                         (I = 0.05)                                                                    Glycine-HCl                                                                              2.5     138.7      ±9.9                                         (I = 0.05)                                                                    Glycine-HCl                                                                              3.0     100.5      ±5.9                                         (I = 0.05)                                                                    Formate    3.5     132.0     ±20.7                                         (I = 0.05)                                                                    Acetate    4.0     209.7     ±12.7                                         (I = 0.05)                                                                    Acetate    5.0     273.1     ±27.7                                         (I = 0.05)                                                                    Acetate    5.5     453.7     ±59.2                                         (I = 0.05)                                                                    ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        K.sub.obs values (1/days) for the degradation of doxorubicin.HCl              2 mg/ml solutions in 0.9% saline at various pHs at 55° C.              Buffer     pH      K.sub.obs × 10.sup.3                                                              95% confidence limits                            ______________________________________                                        Glycine-HCl                                                                              2.0     362.4     ±19.4                                         (I = 0.05)                                                                    Glycine-HCl                                                                              2.5     276.5     ±30.2                                         (I = 0.05)                                                                    Glycine-HCl                                                                              3.0     133.2      ±8.0                                         (I = 0.05)                                                                    Formate    3.5     148.1     ±11.1                                         (I = 0.05)                                                                    Acetate    4.0     215.7     ±35.4                                         (I = 0.05)                                                                    Acetate    5.0     301.2     ±60.1                                         (I = 0.05)                                                                    Acetate    5.5     430.3     ±59.9                                         (I = 0.05)                                                                    ______________________________________                                    

Shelf-life (t 90%) forecast of doxorubicin.HCl 2 mg/ml sterile watersolution adjusted to pH 3.0

5.0 ml of doxorubicin.HCl 2 mg/ml aqueous solution adjusted to pH 3.0with 0.5N HCl were stored, in glass vials, at:

a) 55° C. for 21 days, b) 45° C. and 35° C. for 28 days, c) 27° C. for90 days.

At prefixed times the vials were analyzed for doxorubicin.HCl assay andpH.

The logarithmic plots of the residual concentration versus time werelinear and indicated the degradation of the drug to follow pseudo-firstorder kinetics at constant pH and temperature.

The observed rate constants (K_(obs)) for the degradation werecalculated again by linear regression analysis of a plot of the naturallogarithm of the residual concentration of doxorubicin.HCl (|Dx|_(t))versus time as depicted by the equation previously reported:

    ln|Dx|.sub.t =ln|Dx|.sub.o -K.sub.obs ·t

The Arrhenius equation for the degradation process was calculated fromthe k_(obs) obtained from the different temperatures taken in accountfor the testing (table 11).

Applying the equation, the rate constants for the pseudo-first orderreactions at 4° C., 8° C., 15° C. and 27° C. were calculated, togetherwith the expected t_(90%) at these temperatures.

Table 10 gives the doxorubicin.HCl residual concentration and percentstability at pH 3.0, at different temperatures and times of storage.

FIG. 4 gives the logarithm of the remaining doxorubicin.HClconcentration versus time at different temperatures.

The t_(90%) forecasts (table 11) show that a commercially meaningfulshelf-life can be attributed to doxorubicin.HCl 2 mg/ml pH 3.0 aqueoussolution only if the product is stored in a refrigerator (between 2° C.and 8° C.).

                                      TABLE 10                                    __________________________________________________________________________    Accelerated stability data of doxorubicin.HCl 2 mg/ml pH 3.0 solutions in     sterile water                                                                 at different times and temperatures                                           Storage            Time (days)                                                temperature                                                                          Tests       0   4   8   14  21  28  60  90                             __________________________________________________________________________    27° C.                                                                        Doxorubicin.HCl assay                                                         mg/ml       1.992   1.993                                                                             1.988                                                                             1.962                                                                             1.941                                                                             1.908                                                                             1.850                                 % stability 100.0   100.1                                                                             99.8                                                                              98.5                                                                              97.4                                                                              95.8                                                                              92.9                                  pH          3.00    2.95                                                                              2.94                                                                              2.95                                                                              2.94                                                                              2.96                                                                              2.93                           35° C.                                                                        Doxorubicin.HCl assay                                                         mg/ml       1.992                                                                             1.985                                                                             1.952                                                                             1.889                                                                             1.876                                                                             1.808                                         % stability 100.0                                                                             99.6                                                                              98.0                                                                              94.8                                                                              94.2                                                                              90.8                                          pH          3.00                                                                              2.96                                                                              2.98                                                                              2.93                                                                              2.92                                                                              2.92                                   45° C.                                                                        Doxorubicin.HCl assay                                                         mg/ml       1.992                                                                             1.919                                                                             1.851                                                                             1.677                                                                             1.565                                                                             1.393                                         % stability 100.0                                                                             96.3                                                                              92.9                                                                              84.2                                                                              78.6                                                                              69.9                                          pH          3.00                                                                              2.97                                                                              2.95                                                                              2.85                                                                              2.92                                                                              2.90                                   55° C.                                                                        Doxorubicin.HCl assay                                                         mg/ml       1.992                                                                             1.760                                                                             1.493                                                                             1.036                                                                             0.730                                             % stability 100.0                                                                             88.4                                                                              74.9                                                                              52.0                                                                              36.6                                              pH          3.00                                                                              2.94                                                                              2.90                                                                              2.80                                                                              2.82                                       __________________________________________________________________________

                  TABLE 11                                                        ______________________________________                                        Arrhenius approach. Pseudo-first order rate constants,                        Arrhenius equation, calculated t.sub.90%                                      ______________________________________                                        PSEUDO-FIRST ORDER RATE CONSTANTS,                                            OBSERVED VALUES (K.sub.obs)                                                   Temperature                                                                            K.sub.obs × 10.sup.3 (1/days)                                                          Correlation Coefficient                               ______________________________________                                        27° C.                                                                          0.850          0.986                                                 35° C.                                                                          3.506          0.983                                                 45° C.                                                                          12.790         0.995                                                 55° C.                                                                          49.340         0.995                                                 ______________________________________                                        ARRHENIUS EQUATION FROM                                                       27° C., 35° C., 45° C. AND 55° C. RATE            CONSTANTS                                                                      ##STR1##                                                                     correlation coefficient = 0.9988                                              PSEUDO-FIRST ORDER RATE CONSTANTS,                                            CALCULATED VALUES (K)                                                                                        95%                                            Temperature                                                                            K · 10.sup.3 (1/days)                                                            t.sub.90% (days)                                                                        confidence limits                              ______________________________________                                         4° C.                                                                          0.019       5,652      3,079-10,380                                   8° C.                                                                          0.038       2,745     1,603-4,697                                    15° C.                                                                          0.130         810       532-1,238                                    27° C.                                                                          0.918         115      89-147                                        ______________________________________                                    

Shelf-life (t 90%) forecast of doxorubicin.HCl 2 mg/ml 0.9% sodiumchloride solution adjusted to pH 3.0

5.0 ml of doxorubicin.HCl 2 mg/ml solution in 0.9% sodium chlorideadjusted to pH 3.0 with 0.5N HCl were stored, in glass vials, at: a) 60°C. for 4 days, b) 55° C. for 14 days, c) and 45° C. for 21 days, d) 35°C. for 28 days.

At prefixed times the vials were analyzed for doxorubicin.HCl assay andpH.

The logarithmic plots of the residual concentration versus time werelinear and indicated the degradation of the drug to follow pseudo-firstorder kinetics at constant pH and temperature.

The observed rate constants (K_(obs)) for the degradation werecalculated again by linear regression analysis of a plot of the naturallogarithm of the residual concentration of doxorubicin.HCl (|Dx|_(t))versus time as depicted by the equation previously reported:

    ln|Dx|.sub.t =ln|Dx|.sub.o -K.sub.obs ·t

The Arrhenius equation for the degradation process was calculated fromthe K_(obs) obtained from the different temperature taken in account forthe testing (table 13).

Applying the equation, the rate constants for the pseudo-first orderreactions at 4° C., 8° C., 15° C. and 27° C. were calculated, togetherwith the expected t_(90%) at these temperatures.

Table 12 gives the doxorubicin.HCl residual concentration and percentstability at pH 3.0, at different temperatures and times of storage.

FIG. 5 gives the logarithm of the remaining doxorubicin.HClconcentration versus time at different temperatures.

The t_(90%) forecasts (table 13) show that a commercially meaningfulshelf-life can be attributed to doxorubicin HCl 2 mg/ml pH 3.0 solutionin 0.9% sodium chloride only if the product is stored in a refrigerator(between 2° C. and 8° C.).

                                      TABLE 12                                    __________________________________________________________________________    Accelerated stability data of doxorubicin.HCl 2 mg/ml pH 0.9% soldium         chloride                                                                      at different times and temperatures                                           Storage           Time (days)                                                 Conditions                                                                          Tests       0   1   2   3   4   8   11  14  21  28                      __________________________________________________________________________    35° C.                                                                       Doxorubicin.HCl assay                                                         mg/ml       2.061               2.045   1.946                                                                             1.932                                                                             1.852                         % stability 100.0               99.2    94.4                                                                              93.7                                                                              89.9                          pH          3.05                2.98    2.92                                                                              2.92                                                                              2.98                    45° C.                                                                       Doxorubicin.HCl assay                                                         mg/ml       2.061           1.996                                                                             1.724   1.517                                                                             1.344                             % stability 100.0           96.5                                                                              83.6    73.6                                                                              65.2                              pH          3.05            2.98                                                                              2.97    2.98                                                                              2.93                        55° C.                                                                       Doxorubicin.HCl assay                                                         mg/ml       2.061           1.450                                                                             1.066                                                                             0.900                                     % stability 100.0           70.4                                                                              51.7                                                                              43.7                                      pH          3.05            2.90                                                                              2.97                                                                              2.95                                60° C.                                                                       Doxorubicin.HCl assay                                                         mg/ml       2.061                                                                             1.742                                                                             1.481                                                                             1.290                                                                             1.050                                             % stability 100.0                                                                             84.5                                                                              71.9                                                                              62.6                                                                              50.9                                              pH          3.05                                                                              2.97                                                                              2.96                                                                              2.98                                                                              2.96                                        __________________________________________________________________________

                  TABLE 13                                                        ______________________________________                                        Doxorubicin.HCl 2 mg/ml pH 3.0 solution in 0.9% NaCl                          Arrhenius approach. Pseudo-first order rate constants,                        Arrhenius equation, calculated t.sub.90%                                      ______________________________________                                        PSEUDO-FIRST ORDER RATE CONSTANTS                                             OBSERVED VALUES (K.sub.obs)                                                   Temperature                                                                            K.sub.obs × 10.sup.3 (1/days)                                                          Correlation Coefficient                               ______________________________________                                        35° C.                                                                          3.89           0.965                                                 45° C.                                                                          21.61          0.987                                                 55° C.                                                                          75.90          0.996                                                 60° C.                                                                          164.90         0.998                                                 ______________________________________                                        ARRHENIUS EQUATION FROM                                                       35° C., 45° C., 55° C. AND 60° C. RATE            CONSTANTS                                                                      ##STR2##                                                                     correlation coefficient = 0.9986                                              PSEUDO-FIRST ORDER RATE CONSTANTS,                                            CALCULATED VALUES (K)                                                         Tem-                           95%                                            perature                                                                              K × 10.sup.3 (1/days)                                                                t.sub.90% (days)                                                                        confidence limits                              ______________________________________                                         4° C.                                                                         0.017        6,166      1,670-22,756                                   8° C.                                                                         0.037        2,838       861-9,351                                    15° C.                                                                         0.137          768       281-2,105                                    27° C.                                                                         1.112          94       45-197                                        ______________________________________                                    

Shelf-life (t 90%) forecast of doxorubicin.HCl 2 mg/ml solution in 5%dextrose adjusted to pH 3.0

5.0 ml of doxorubicin.HCl 2 mg/ml solution in 5% dextrose adjusted to pH3.0 with 0.5N HCl were stored, in glass vials, at: a) 60° C. for 8 days,b) 55° C. for 17 days, c) 45° C. and 35° C. for 28 days.

At prefixed times the vials were analyzed for doxorubicin.HCl assay andpH.

The logarithmic plots of the residual concentration versus time werelinear and indicated the degradation of the drug to follow pseudo-firstorder kinetics at constant pH and temperature. The observed rateconstants (K_(obs)) for the degradation was calculated again by linearregression analysis of a plot of the natural logarithm of the residualconcentration of doxorubicin.HCl (|Dx|_(t)) versus time as depicted bythe equation previously reported:

    ln |Dx|.sub.t =ln |Dx|.sub.o -K.sub.obs ·t

The Arrhenius equation for the degradation process was calculated fromthe K_(obs) obtained from the different temperature taken in account forthe testing (table 15).

Applying the equation, the rate constants for the pseudo-order reactionsat 4° C., 8° C., and 15° C. and 27° C. were calculated, together withexpected t_(90%) at these temperatures.

Table 14 gives the doxorubicin.HCl residual concentration and percentstability at pH 3.0, at different temperatures and times of storage.

FIG. 6 gives the logarithm of the remaining doxorubicin.HClconcentration versus time at different temperatures.

The t₉₀ % forecasts (table 15) show that a commercially meaningfulshelf-life can be attributed to doxorubicin.HCl 2 mg/ml pH 3.0 5%dextrose solution only if the product is stored in a refrigerator(between 2° C. and 8° C.).

                                      TABLE 14                                    __________________________________________________________________________    Accelerated stability data of doxorubicin.HCl 2 mg/ml pH 3.0 solution in      5% dextrose                                                                   at different times and temperatures                                           Storage           Time (days)                                                 Conditions                                                                          Tests       0   2   4   6   8   11  14  17  21  28                      __________________________________________________________________________    35° C.                                                                       Doxorubicin.HCl assay                                                         mg/ml       2.114   2.044   2.034   2.015   1.934                                                                             1.859                         % stability 100.0   96.7    96.2    53.3    91.5                                                                              87.9                          pH          3.02    2.98    2.94    2.95    2.90                                                                              2.94                    45° C.                                                                       Doxorubicin.HCl assay                                                         mg/ml       2.114   1.940   1.870   1.684   1.510                                                                             1.410                         % stability 100.0   91.8    88.5    79.7    71.5                                                                              66.7                          pH          3.02    2.97    2.98    2.95    2.96                                                                              2.96                    55° C.                                                                       Doxorubicin.HCl assay                                                         mg/ml       2.114   1.718   1.415   1.112   0.957                                                                             0.796                         % stability 100.0   81.3    66.9    52.6    45.3                                                                              37.7                          pH          3.02    2.95    2.92    2.99    2.91                                                                              2.95                    60° C.                                                                       Doxorubicin.HCl assay                                                         mg/ml       2.114                                                                             1.752                                                                             1.393                                                                             1.176                                                                             0.925                                             % stability 100.0                                                                             82.9                                                                              65.9                                                                              55.7                                                                              43.8                                              pH          3.02                                                                              2.96                                                                              2.98                                                                              2.96                                                                              2.97                                        __________________________________________________________________________

                  TABLE 15                                                        ______________________________________                                        Doxorubicin.HCl 2 mg/ml pH 3.0 solution in 5% dextrose.                       Arrhenius approach. Pseudo-first order rate constants,                        Arrhenius equation, calculated t.sub.90%                                      ______________________________________                                        PSEUDO-FIRST ORDER RATE CONSTANTS                                             OBSERVED VALUES (K.sub.obs)                                                   Temperature                                                                            K.sub.obs × 10.sup.3 (1/days)                                                          Correlation Coefficient                               ______________________________________                                        35° C.                                                                          4.190          0.990                                                 45° C.                                                                          14.55          0.995                                                 55° C.                                                                          58.11          0.998                                                 60° C.                                                                          102.6          0.999                                                 ______________________________________                                        ARRHENIUS EQUATION FROM                                                       35° C., 45° C., 55° C. AND 60° C. RATE            CONSTANTS                                                                      ##STR3##                                                                     correlation coefficient = 0.9993                                              PSEUDO-FIRST ORDER RATE CONSTANTS,                                            CALCULATED VALUES (K)                                                         Tem-                           95%                                            perature                                                                              K × 10.sup.3 (1/days)                                                                t.sub.90% (days)                                                                        confidence limits                              ______________________________________                                         4° C.                                                                         0.0326       3,218     1,463-7,082                                     8° C.                                                                         0.0645       1,628       792-3,344                                    15° C.                                                                         0.203          516     281-949                                        27° C.                                                                         1.283          82       53-128                                        ______________________________________                                    

Long term stability of doxorubicin formulations having a pH fallingwithin the range from 2.5 to 3.5

Batches tested, formulations tested and packaging used are reported on,respectively, tables 16, 17 and 18, as well as on, respectively, tables25, 26 and 27.

Test and methods

The formulations were tested as regards appearance, clarity of solution,pH, sterility (8° C., yearly), doxorubicin.HCl assay.

Test methods

For appearance and clarity; visual inspection

For pH: USP XXI

For sterility: USP XXI (membrane filtration)

For doxorubicin.HCl assay: HPLC ion-pair method and USP HPLC method (USPXXI)

Brief description of the HPLC ion-pair method for doxorubicin.

    ______________________________________                                        HCl assay:                                                                    ______________________________________                                        Column filling   reverse phase, Zorbax TMS                                    Mobile phase     water, acetonitrile,                                                          methanol (54:29:17 v/v/v)                                                     containing 2 ml/l 85%                                                         phosphoric acid and 1 mg/ml                                                   sodium laurylsulfate                                                          (pairing agent) adjusted to                                                   pH 3.5 with 2N NaOH                                          Mobile phase flow rate                                                                         1.5 ml/min                                                   Column temperature                                                                             ambient (22° C. ± 2° C.)                    Analytical wavelength                                                                          254 nm                                                       System suitability                                                                             Symmetry factor between 0.7                                  parameters       and 1.2; number of                                                            theoretical plates ≧2500;                                              measurement reproducibility:                                                  variation coefficient <1,                                                     n = 6; resolution factor ≧12                          ______________________________________                                    

The HPLC ion-pair method for doxorubicin.HCl assay is validated foraccuracy, precision, linearity, sensitivity, specifically andstability-indicating nature.

The results obtained for:

percent doxorubicin.HCl stability (ion-pair method) and

pH

referred to the vials stored in upright position are given in:

Table 19 storage at -20°

Tables 20 and 28 storage at +4° C.

Tables 21 and 29 storage at +8° C.

Tables 22 and 30 storage at +15° C.

Tables 23 and 31 storage at +27° C.

Table 24 storage at 100 and 250 foot candles

Table 32 storage at 250 foot candles.

The doxorubicin.HCl assays given in these tables are the mean of threeindependent determinations.

As far as the other parameters checked during stability:

the clarity of the solution was unchanged at all the checks carried outat all the storage conditions applied;

the appearance of the solutions was: a) unchanged at all the checkscarried out on samples stored at 4° C. and 8° C., b) slightly darkenedafter: 9 months at 15° C., 3 months at 27° C., 3 months at 100 and 250foot candles light;

the closure system was unchanged at all the checks carried out at allthe storage conditions;

the sterility was maintained after 18 months at 8° C.

The results of the controls carried out on the vials stored in invertedposition do not differ significantly from those on the vials in uprightposition.

The percent doxorubicin.HCl stability values obtained by the USP HPLCmethod do not differ significantly from those obtained by the HPLCion-pair method given in Tables 19-24.

The obtained stability data indicate that the tested doxorubicin.HClsolutions having different pH values within the range from 2.5 to 3.5reach the lower limit of acceptance (90% of nominal concentration) inabout 9 and 2-3 months at 15° C. and, respectively 27° C., but provestable up to 18 months at 4° C. and 8° C., i.e. at temperature usuallyadopted for the storage of the products under refrigeration.

In distinct contrast, the doxorubicin.HCl solution obtained uponreconstitution of the commercial freeze-dried preparate, whose pH variesbetween 4.5 and 6, shows a much lower degree of stability as shown bythe fact that it is recommended to discard reconstituted solutions afteronly 48 hours storage in refrigerator according to the leafletaccompanying packages of Adriamycin (i.e. doxorubicin.HCl) in the UnitedStates.

                                      TABLE 16                                    __________________________________________________________________________    Stability studies, Batches tested.                                            Batch    Batch No.                                                            Characteristics                                                                        TF/23049                                                                            TF/23077                                                                            TF/23078                                                                            TF/23117                                                                            TF/23119                                                                            H0001 L0001 M0001                      __________________________________________________________________________    Doxorubicin.HCl                                                                        10    10    10    20    50    10    20    50                         per vial (mg)                                                                 pH       3.06  2.81  3.50  2.97  3.08  3.15  3.05  3.20                       Formulation No.                                                                        F16804/IL1                                                                          F16804/IL1                                                                          F16804/IL1                                                                          F16804/IL2                                                                          F16804/IL3                                                                          F16804/IL4                                                                          F16804/IL5                                                                          F16804/IL6                 Batch size                                                                             700   400   400   500   500   2,400 2,300 2,400                      No. of vials                                                                  __________________________________________________________________________

                                      TABLE 17                                    __________________________________________________________________________    Stability studies. Formulations tested.                                       Composition per                                                                        Formulation number                                                   vial     FI6804/IL1                                                                          FI6804/IL2                                                                          FI6804/IL3                                                                          FI6804/IL4                                                                          FI6804/IL5                                                                          FI6804/IL6                             __________________________________________________________________________    Doxorubicin.HCl                                                                        10    20    50    10    20    50                                     mg                                                                            Hydrochloric acid                                                                      2.8-3.5                                                                             2.8-3.5                                                                             2.8-.35                                                                             2.8-3.5                                                                             2.8-3.5                                                                             2.8-3.5                                q.s. to pH                                                                    Water q.s. to ml                                                                       5.0   10.0  25.0  5.0   10.0  25.0                                   __________________________________________________________________________

                                      TABLE 18                                    __________________________________________________________________________    Stability studies. Packaging used.                                                    Batch No.                                                             Packaging                                                                             TF/23049                                                                            TF/23077                                                                            TF/23078                                                                            TF/231117                                                                           TF/23119                                                                            H0001 L0001 M0001                       __________________________________________________________________________    vial glass type                                                                       I     I     I     I     I     I     I     I                           vial top                                                                              8 ml  8 ml  8 ml  14 ml 39 ml 10 ml 14 ml 39 ml                       capacity                                                                      stopper chlorobutyl                                                                         chlorobutyl                                                                         chlorobutyl                                                                         chlorobutyl                                                                         chlorobutyl                                                                         chlorobutyl                                                                         chlorobutyl                                                                         chlorobutyl                         rubber,                                                                             rubber,                                                                             rubber,                                                                             rubber,                                                                             rubber,                                                                             rubber,                                                                             rubber,                                                                             rubber,                             teflon-faced                                                                        teflon-faced                                                                        teflon-faced                                                                        teflon-faced                                                                        teflon-faced                                                                        teflon-faced                                                                        teflon-faced                                                                        teflon-faced                seal    aluminum                                                                            aluminum                                                                            aluminum                                                                            aluminum                                                                            aluminum                                                                            aluminum                                                                            aluminum                                                                            aluminum                    __________________________________________________________________________

                  TABLE 19                                                        ______________________________________                                        Doxorubicin.HCl 2 mg/ml solution. Stability data at                           -20° C. (vials stored upright) acquired up to 3 months.                Batch                Time - Months                                            Dosage               0         1     3                                        ______________________________________                                        H0001   doxorubicin.HCl                                                                            100       99.9  99.6                                     10 mg   % stability                                                                   pH           3.15      3.12  2.98                                     L0001   doxorubicin.HCl                                                                            100       100.8 99.8                                     20 mg   % stability                                                                   pH           3.05      2.84  2.97                                     M0001   doxorubicin.HCl                                                                            100       100.7 101.0                                    50 mg   % stability                                                                   pH           3.20      2.96  2.99                                     ______________________________________                                    

                  TABLE 20                                                        ______________________________________                                        Doxorubicin.HCl 2 mg/ml solution.                                             Stability data at 4° C. (vials stored upright)                         acquired up to 18 months                                                      Batch         Time Months                                                     Dosage        0      1     3     6    9    12   18                            ______________________________________                                        TF/23049                                                                              *     100    99.9  100.6 98.3 98.2 97.7 96.9                          10 mg   **    3.06   3.10  3.09  3.10 3.05 2.97 3.07                          TF/23077                                                                              *     100    101.7 99.3  97.9 98.0 99.8                               10 mg   **    2.81   2.86  2.75  2.65 2.67 2.76                               TF/23078                                                                              *     100    101.2 98.8  97.8 98.8 96.8                               10 mg   **    3.50   3.54  3.49  3.44 3.43 3.54                               TF/23117                                                                              *     100    96.8  96.6  98.1 98.8 97.5                               20 mg   **    2.97   2.98  2.92  2.86 2.95 2.98                               TF/23119                                                                              *     100    98.6  99.1  98.9 98.4 97.5                               50 mg   **    3.08   2.98  2.98  2.89 2.99 3.00                               H0001   *     100          97.6  99.2                                         10 mg   **    3.15   n.d.  3.06  3.22                                         L0001   *     100          98.8  98.4                                         20 mg   **    3.05   n.d.  2.99  2.94                                         M0001   *     100          99.7  99.7                                         50 mg   **    3.20   n.d.  3.00  3.04                                         ______________________________________                                         * doxorubicin.HCl % stability                                                 ** pH                                                                         n.d. = not determined.                                                   

                                      TABLE 21                                    __________________________________________________________________________    Doxoribicin.HCl 2 mg/ml solution.                                             Stability data at 8° C. (vials stored upright)                         acquired up to 18 months.                                                     Batch   Time Months                                                           Dosage  0   1   2   3   6   9   12  18                                        __________________________________________________________________________    TF/23049                                                                            * 100 99.7    100.1                                                                             96.5                                                                              96.1                                                                              96.5                                                                              95.4                                      10 mg **                                                                              3.06                                                                              3.07    3.09                                                                              3.07                                                                              3.04                                                                              2.96                                                                              3.04                                      TF/23077                                                                            * 100 102.1   101.6                                                                             97.5                                                                              96.6                                                                              95.0                                          10 mg **                                                                              2.81                                                                              2.81    2.74                                                                              2.65                                                                              2.67                                                                              2.75                                          TF/23078                                                                            * 100 98.3    97.7                                                                              96.5                                                                              95.9                                                                              98.8                                          10 mg **                                                                              3.50                                                                              3.59    3.47                                                                              3.27                                                                              3.43                                                                              3.51                                          TF/23117                                                                            * 100 95.7    95.8                                                                              97.8                                                                              96.2                                                                              95.5                                          20 mg **                                                                              2.97                                                                              2.97    2.92                                                                              2.85                                                                              2.96                                                                              2.98                                          TF/23119                                                                            * 100 97.6    97.8                                                                              96.2                                                                              97.3                                                                              96.8                                          50 mg **                                                                              3.08                                                                              2.94    2.94                                                                              2.87                                                                              2.99                                                                              3.00                                          H0001 * 100 98.2                                                                              99.4                                                                              96.4                                                                              96.7                                                  10 mg **                                                                              3.15                                                                              3.12                                                                              3.16                                                                              3.05                                                                              3.23                                                  L0001 * 100 100.6                                                                             99.1                                                                              98.1                                                                              98.3                                                  20 mg **                                                                              3.05                                                                              2.84                                                                              2.83                                                                              2.97                                                                              2.94                                                  M0001 * 100 100.3                                                                             100.6                                                                             98.7                                                                              99.0                                                  50 mg **                                                                              3.20                                                                              2.96                                                                              2.97                                                                              3.01                                                                              3.03                                                  __________________________________________________________________________     * doxorubicin.HCl % stability                                                 ** pH                                                                    

                                      TABLE 22                                    __________________________________________________________________________    Doxorubicin.HCl 2 mg/ml solution. -Stability data at 15° C. (vials     stored upright)                                                               acquired up to 12 months.                                                     Batch   Time Months                                                           Dosage  0   0.5 1   2   3   6   9   12                                        __________________________________________________________________________    TF/23049                                                                            * 100 97.8                                                                              98.9    97.1                                                                              92.7                                                                              92.9                                                                              90.2                                      10 mg **                                                                              3.06                                                                              3.03                                                                              3.07    3.10                                                                              3.08                                                                              3.02                                                                              2.95                                      TF/23077                                                                            * 100 100.4                                                                             101.9   98.8                                                                              94.6                                                                              92.7                                                                              91.1                                      10 mg **                                                                              2.81                                                                              2.81                                                                              2.85    2.71                                                                              2.63                                                                              2.67                                                                              2.74                                      TF/23078                                                                            * 100 101.4                                                                             98.4    95.3                                                                              94.6                                                                              91.9                                                                              90.7                                      10 mg **                                                                              3.50                                                                              3.51                                                                              3.58    3.47                                                                              3.38                                                                              3.41                                                                              3.47                                      TF/23117                                                                            * 100 99.1                                                                              96.4    95.2                                                                              94.6                                                                              90.7                                          20 mg **                                                                              2.97                                                                              2.95                                                                              2.95    2.90                                                                              2.81                                                                              2.95                                          TF/23119                                                                            * 100 97.4                                                                              97.1    95.9                                                                              92.7                                                                              90.6                                          50 mg **                                                                              3.08                                                                              2.99                                                                              2.95    2.91                                                                              2.87                                                                              2.98                                          H0001 * 100     97.9                                                                              97.1                                                                              94.8                                                                              94.6                                              10 mg **                                                                              3.15    3.12                                                                              3.16                                                                              3.06                                                                              3.23                                              L0001 * 100     100.5                                                                             98.7                                                                              96.3                                                                              95.5                                              20 mg **                                                                              3.05    2.85                                                                              2.87                                                                              2.98                                                                              2.96                                              M0001 * 100     99.4                                                                              100.3                                                                             97.2                                                                              95.6                                              50 mg **                                                                              3.20    2.96                                                                              2.94                                                                              3.01                                                                              3.04                                              __________________________________________________________________________     * doxorubicin.HCl % stability                                                 ** pH                                                                    

                  TABLE 23                                                        ______________________________________                                        Doxorubicin.HCl 2 mg/ml solution. -Stability data at 27° C. (vials     stored upright)                                                               acquired up to 6 months.                                                      Batch         Time Months                                                     Dosage        0       0.5   1     2    3     6                                ______________________________________                                        TF/23049                                                                              *     100     97.2  95.8       87.9  73.6                             10 mg   **    3.06    2.98  3.07       3.08  3.03                             TF/23077                                                                              *     100     98.5  96.2       86.4  69.2                             10 mg   **    2.81    2.80  2.85       2.71  2.64                             TF/23078                                                                              *     100     101.2 94.5       80.5  71.1                             10 mg   **    3.50    3.51  3.58       3.38  3.13                             TF/23117                                                                              *     100     97.4  93.2       81.9  66.6                             20 mg   **    2.97    2.95  2.94       2.88  2.77                             TF/23119                                                                              *     100     96.0  93.3       85.3  66.8                             50 mg   **    3.08    2.97  2.97       2.91  2.82                             H0001   *     100           94.5  94.2 86.6                                   10 mg   **    3.15          3.10  3.09 3.01                                   L0001   *     100           97.2  94.3 89.3                                   20 mg   **    3.05          2.84  2.85 2.96                                   M0001   *     100           96.5  93.6 88.1                                   50 mg   **    3.20          2.95  2.95 2.99                                   ______________________________________                                         * doxorubicin.HCl % stability                                                 ** pH                                                                    

                                      TABLE 24                                    __________________________________________________________________________    Doxorubicin.HCl 2 mg/ml solution. Stability data at 100                       and 250 f.c. (vials stored inverted) acquired up to 3                         months.                                                                               Time Months                                                           Batch   100 foot-candles                                                                              250 foot-candles                                      Dosage  0   0.5 1   3   0.5 1   2   3                                         __________________________________________________________________________    TF/23049                                                                            * 100 96.3                                                                              95.9                                                                              81.3                                                                              95.9                                                                              94.8                                              10 mg **                                                                              3.06                                                                              3.05                                                                              3.05                                                                              3.06                                                                              2.99                                                                              3.04                                              TF/23077                                                                            * 100 98.3                                                                              98.1                                                                              87.7                                                                              97.3                                                                              94.5                                              10 mg **                                                                              2.81                                                                              2.79                                                                              2.84                                                                              2.70                                                                              2.79                                                                              2.84                                              TF/23078                                                                            * 100 99.6                                                                              96.4                                                                              88.0                                                                              97.8                                                                              89.7                                              10 mg **                                                                              3.50                                                                              3.50                                                                              3.58                                                                              3.39                                                                              3.47                                                                              3.53                                              TF/23117                                                                            * 100 96.8                                                                              96.7                                                                              91.7                                                                              98.1                                                                              94.6                                              20 mg **                                                                              2.97                                                                              2.93                                                                              2.95                                                                              2.87                                                                              2.95                                                                              2.93                                              TF/23119                                                                            * 100 96.9                                                                              96.7                                                                              89.6                                                                              96.4                                                                              95.0                                              50 mg **                                                                              3.08                                                                              2.96                                                                              2.95                                                                              2.93                                                                              2.96                                                                              2.97                                              H0001 * 100                 95.2                                                                              93.7                                                                              87.8                                      10 mg **                                                                              3.15                3.10                                                                              3.06                                                                              2.97                                      L0001 * 100                 96.5                                                                              93.0                                                                              86.5                                      20 mg **                                                                              3.05                2.84                                                                              2.85                                                                              2.97                                      M0001 * 100                 97.8                                                                              91.5                                                                              85.3                                      50 mg **                                                                              3.20                2.95                                                                              2.94                                                                              2.99                                      __________________________________________________________________________     * doxorubicin.HCl % stability                                                 ** pH                                                                    

                  TABLE 25                                                        ______________________________________                                        Stability studies. Batches tested.                                            Batch      Batch No.                                                          Characteristics                                                                          P0001       Q0001     R0001                                        ______________________________________                                        Doxorubicin.HCl                                                                            10          20        50                                         per vial (mg)                                                                 pH         3.00        3.00      3.00                                         Formulation No.                                                                          FI6804/IL7  FI6804/IL8                                                                              FI6804/IL9                                   Batch size 2,400       2,200     2,500                                        No. of vials                                                                  ______________________________________                                    

                  TABLE 26                                                        ______________________________________                                        Stability studies. Formulations tested.                                       Composition per                                                                             Formulation number                                              vial          F16804/IL7                                                                              F16804/IL8 F16804/IL9                                 ______________________________________                                        Doxorubicin.HCl                                                                             10        20         50                                         mg                                                                            Hydrochloric acid                                                                           2.8-3.5   2.8-3.5    2.8-3.5                                    q.s. to pH                                                                    0,9% sodium chloride                                                                        5.0       10.0       25.0                                       injection q.s. to ml                                                          ______________________________________                                    

                  TABLE 27                                                        ______________________________________                                        Stability studies. Packaging used.                                                    Batch No.                                                             Packaging P0001        Q0001     R0001                                        ______________________________________                                        vial glass type                                                                         I            I         I                                            vial top  10 ml        14 ml     39 ml                                        capacity                                                                      stopper   chlorobutyl  chlorobutyl                                                                             chlorobutyl                                            rubber,      rubber,   rubber,                                                teflon-faced teflon-faced                                                                            teflon-faced                                 seal      aluminum     aluminum  aluminum                                     ______________________________________                                    

                  TABLE 28                                                        ______________________________________                                        Doxorubicin.HCl 2 mg/ml solution in Saline                                    for Injection at pH = 3. Stability data at                                    4° C. (vials stored upright) acquired up to 9                          and 12 months.                                                                Batch              Time - Months                                              Dosage             0       3   6     9     12                                 ______________________________________                                        P0001 doxorubicin.HCl                                                                            100    98.3 98.0  99.2                                     10 mg % stability                                                                   pH           3.00   2.93 2.98  2.90                                     Q0001 doxorubicin.HCl                                                                            100    97.5 97.0  100.1                                    20 mg % stability                                                                   pH           3.01   3.06 3.03  3.00                                     R0001 doxorubicin.HCl                                                                            100    99.8 100.7 101.2 101.7                              50 mg % stability                                                                   pH           3.02   3.08 3.15  3.14   3.10                              ______________________________________                                    

                                      TABLE 29                                    __________________________________________________________________________    Doxorubicin.HCl 2 mg/ml solution in Saline for Injection                      at pH = 3. Stability data at 8° C. (vials stored upright)              acquired up to 9 and 12 months.                                               Batch        Time - Months                                                    Dosage       0   1   2   3   6  9   12                                        __________________________________________________________________________    P0001                                                                             doxorubicin.HCl                                                                        100 101.0                                                                             100.6                                                                             97.9                                                                              97.4                                                                             96.8                                          10 mg                                                                             % stability                                                                   pH       3.00                                                                              2.93                                                                              2.89                                                                              2.91                                                                              3.00                                                                             2.90                                          Q0001                                                                             doxorubicin.HCl                                                                        100 99.4                                                                              99.9                                                                              96.8                                                                              96.7                                                                             95.7                                          20 mg                                                                             % stability                                                                   pH       3.01                                                                              3.02                                                                              3.01                                                                              3.05                                                                              3.02                                                                             3.00                                          R0001                                                                             doxorubicin.HCl                                                                        100 99.8                                                                              99.8                                                                              98.4                                                                              98.5                                                                             99.5                                                                              100.9                                     50 mg                                                                             % stability                                                                   pH       3.02                                                                              3.02                                                                              3.09                                                                              3.08                                                                              3.13                                                                             3.13                                                                              3.10                                      __________________________________________________________________________

                                      TABLE 30                                    __________________________________________________________________________    Doxorubicin.HCl 2 mg/ml solution in Saline for Injection                      at pH = 3. Stability data at 15° C. (vials stored upright)             acquired up to 9 and 12 months.                                               Batch        Time - Months                                                    Dosage       0   1   2   3   6  9   12                                        __________________________________________________________________________    P0001                                                                             doxorubicin.HCl                                                                        100 100.6                                                                             99.9                                                                              95.9                                                                              94.0                                                                             89.1                                          10 mg                                                                             % stability                                                                   pH       3.00                                                                              2.93                                                                              2.89                                                                              2.90                                                                              2.99                                                                             2.90                                          Q0001                                                                             doxorubicin.HCl                                                                        100 98.6                                                                              97.8                                                                              95.1                                                                              96.4                                                                             89.8                                          20 mg                                                                             % stability                                                                   pH       3.01                                                                              3.01                                                                              3.01                                                                              3.04                                                                              3.01                                                                             3.00                                          R0001                                                                             doxorubicin.HCl                                                                        100 98.8                                                                              97.5                                                                              97.6                                                                              94.7                                                                             96.0                                                                              94.5                                      50 mg                                                                             % stability                                                                   pH       3.02                                                                              3.02                                                                              3.08                                                                              3.08                                                                              3.14                                                                             3.11                                                                              3.10                                      __________________________________________________________________________

                  TABLE 31                                                        ______________________________________                                        Doxorubicin.HCl 2 mg/ml solution in Saline                                    for Injection at pH = 3. Stability data at                                    27° C. (vials stored upright) acquired up to                           3 months.                                                                     Batch                Time - Months                                            Dosage               0        1    2      3                                   ______________________________________                                        P0001   doxorubicin.HCl                                                                            100      98.3 95.0   84.9                                10 mg   % stability                                                                   pH           3.00     2.93 2.89   2.88                                Q0001   doxorubicin.HCl                                                                            100      96.0 93.2   83.8                                20 mg   % stability                                                                   pH           3.01     3.01 2.99   3.03                                R0001   doxorubicin.HCl                                                                            100      95.6 92.2   88.7                                50 mg   % stability                                                                   pH           3.02     3.02 3.06   3.05                                ______________________________________                                    

                  TABLE 32                                                        ______________________________________                                        Doxorubicin.HCl 2 mg/ml solution in Saline                                    for Injection at pH = 3. Stability data at                                    R.T. +250 f.c. (vials stored upright) acquired                                up to 3 months.                                                               Batch                Time - Months                                            Dosage               0        1    2      3                                   ______________________________________                                        P0001   doxorubicin.HCl                                                                            100      89.6 86.5   70.3                                10 mg   % stability                                                                   pH           3.00     2.92 2.86   2.84                                Q0001   doxorubicin.HCl                                                                            100      91.1 84.5   72.7                                20 mg   % stability                                                                   pH           3.01     2.99 2.97   2.98                                R0001   doxorubicin.HCl                                                                            100      96.0 91.4   86.6                                50 mg   % stability                                                                   pH           3.02     3.01 3.04   3.02                                ______________________________________                                    

Stabilizing effect of various agents.

                  TABLE 33                                                        ______________________________________                                        Stability Data of Doxorubicin Solution, 2 mg/ml                               and a pH of 3.0 at 45° C.                                              Stabilizing Agent                                                                             % Initial                                                     and Its Concentration                                                                         1 Wk    2 Wk     4 Wk  8 Wk                                   ______________________________________                                        Water           87.8    75.9     53.8  25.5                                   5% Dextrose     91.1    82.3     65.6  38.8                                   5% Galactose    91.5    86.1     64.3  --                                     5% Fructose     91.9    80.6     64.1  --                                     4% α-L(-)-Fucose                                                                        91.2    81.9     63.8  --                                     4% α-D(+)-Fucose                                                                        91.8    81.9     63.3  --                                     1% Lactose      91.3    81.7     64.5  34.8                                   4% Dextran, MW 9,000                                                                          90.5    81.5     --    --                                     4% Dextran, MW 506,000                                                                        92.0    84.0     --    --                                     4% α-Cyclodextrin                                                                       91.7    84.3     --    --                                     4% β-Cyclodextrin                                                                        92.1    84.1     --    --                                     4% γ-Cyclodextrin                                                                       94.3    89.0     --    --                                     5% Mannitol     90.7    81.4     65.8  41.1                                   5% Sorbitol     91.4    83.0     67.2  42.5                                   0.5% Thioglycerol                                                                             90.8    83.2     63.5  --                                     5% Inositol     91.7    84.9     --    --                                     5% Ethanol      92.2    85.6     --    --                                     10% Glycerol    92.2    83.4     65.5  --                                     ______________________________________                                         Note: The same stabilizing effect may be seen for the above agents at         lower concentrations, e.g., lower by up to 25-50 wt. %.                  

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A sealed container containing therein a stable,injectable, sterile, pyrogen-free doxorubicin anti-tumor composition ina solution which consists essentially of doxorubicin hydrochloridedissolved in a physiologically acceptable solvent therefor, wherein saidsolution has not been reconstituted from lyophilizate, and wherein saidsolution has a pH adjusted to from 2.5-3.5 with hydrochloric acid and aconcentration of said doxorubicin is from 0.1 to 100 mg/ml.
 2. Acontainer according to claim 1, wherein said container is hermeticallysealed.
 3. A container according to claim 1, wherein the pH of saidsolution is from 2.7 to 3.5.
 4. A container according to claim 1,wherein said physiologically acceptable solvent is a physiologicallyacceptable saline solution.
 5. A container according to claim 1, whereinthe pH of said solution is from 2.7 up to 3.0.
 6. A container accordingto claim 1, wherein said physiologically acceptable solvent is aphysiologically acceptable dextrose solution.
 7. A container accordingto claim 4, wherein said physiologically acceptable solvent is sterilewater.
 8. A container according to claim 1, wherein the physiologicallyacceptable solvent is selected from the group consisting of water,ethanol, polyethylene glycol, dimethylacetamide and mixture thereof. 9.A container according to claim 1, which further contains a tonicityadjustment agent selected from the group consisting of sodium chloride,dextrose, lactose, mannitol, and sorbitol.
 10. A container according toclaim 9, containing about 0.9% w/v sodium chloride.
 11. A containeraccording to claim 9, containing about 5% w/v dextrose, lactose,mannitol or sorbitol.
 12. A container according to claim 1, whichfurther contains a preservative selected from the group consisting ofesters of para-hydroxybenzoic acid and chlorocresol.
 13. A containeraccording to claim 1, wherein the concentration of doxorubicin is from0.1 mg/ml to 50 mg/ml.
 14. A container according to claim 1, wherein theconcentration of doxorubicin is from 1 mg/ml to 20 mg/ml.
 15. Acontainer according to claim 1, wherein the rate constant ofdecomposition, measured under accelerated conditions at a temperature ofaround 55° C. and a concentration of said doxorubicin as thehydrochloride of around 2 mg/ml, is less than about 0.150 day¹.